The discovery that poly(Lysine)(poly(Lys)) can serve as carrier for methotrexate (MTX) and can overcome drug resistance due to defective drug transport in cultured cells is being extended to other carriers and other drugs. Because MTX-poly(Lys) must be degraded in the cells in order to act, it will serve as a tool to study the steps and factors involved in the membrane transport, intracellular traffic, and lysosomal processing of foreign macromolecules. This analysis will be helped by the isolation and characterization of mutants resistant to MTX-poly(Lys) but sensitive to MTX. MTX-poly-((Lys) may enter cells differently when it is complexed with heparin and may inhibit cytoplasmi dihydrofolate reductase without prior processing. This would imply that the intact complex crosses either the lysosomal or the plasma membrane directly, a postulate that will be tested. If such is the case, the heparin complex of MTX-poly-(Lys) will be used to investigate alternate pathways in the membrane transport of macromolecules. The variables to be investigated for their possible influence on the intracellular activation of MITX-poly(Lys) and the transport of its heparin complex include: the transformed phenotype of temperature sensitive mutants, the malignant state of cultured cells, the external and lysosomal pH, the temperature during the transport phase and during the processing phase, cell shape, other factors affecting cytoskeletal organization, inhibitors of lysosomal proteases. Also to be investigated are the reasons why an excess of free poly(Lys) inhibits the cytocidal effect of MTX-poly(Lys), why the intracellular half-life of poly(Lys) is longer than that of average proteins, and whether intracellular poly(Lys) is recycling to the cell surface. This research carried out on cultured cells will require cell-fractiionation, gel filtration and electrophoresis, immuno-cytochemistry and electron microscopy. The questions asked relate to mammalian cell genetics, cell biology, cell pharmacology, experimental cancer chemotherapy, and cancer biology. Defects in the transport and processing of macromolecules have been associated with several important genetic diseases, and the use of poly(Lys) as a drug carrier has potential value for cancer chemotherapy.